The main goal of this project is to demonstrate the feasibility of combining novel cancer testing using Frster resonance energy transfer (FRET) with an emerging platform for clinical diagnostics. The intent is to develop an assay capable of detecting and monitoring bladder cancer (BlCa) using a urine sample. The assay targets a family of matrix metalloproteinases (MMPs) and hyaluronidase (HA-ase). The presence and relative distribution of these enzyme markers is strongly correlated with progression of BlCa. The principle of the assay involves the preparation of immobilized peptide substrates that are dual-labeled with fluorescent dyes and the determination of the rate at which they are cleaved by MMPs or HA-ase present in a patient sample. This will be done by measuring the extent of FRET between the two fluorescent moieties linked through the target substrates for each respective MMPs or HA-ase. In order to differentiate between sequences, each substrate will be immobilized on different p-Chips, which are ultra-small integrated circuits with unique serial numbers. Invented by PharmaSeq, Inc., each p-Chip's distinctive identification number (ID) is transmitted to an analyzer when the p-Chip is illuminated with laser light. The association of a specific substrate with an ID allows construction of a p-Chip-mounted database in which the identity of a target can be determined by the ID of the p-Chip on which it resides. Multiple p-Chips carrying different substrates for different enzymes can be mixed together in a single assay (multiplexing). Results are derived by sequentially measuring the fluorescence and obtaining the ID of each p-Chip in the assay. This is accomplished by passing the p-Chips through a flow-based reader (Simuplex, also manufactured by PharmaSeq). Sensitivity of the assay can be increased using modified p-Chips onto which a silver nanoparticle film has been deposited prior to peptide-dye conjugation. The benefit of the proposed work is the creation of a highly sensitive multiplex, enzymatic assay capable of simultaneously detecting low concentrations of several enzyme biomarkers that are indicative of cancer. The research plan involves 1) an evaluation of the suitability of the silver nanoparticle film prepared on the p-Chip for maximum fluorescence enhancement, and 2) the application of the selected silver nanoparticle configuration toward enhanced detection of metastasis markers for bladder cancer. We will use the capability of the PharmaSeq Simuplex flow reader to simultaneously excite and detect fluorescence at two wavelengths suited for two dyes, Cy3 and Cy5, and evaluate the FRET ratio changes resulting from the release of a target fragment in the substrate cleaving process. Our final goal is to take advantage of the metal-fluorophore interactions and develop a highly sensitive assay on the p-Chip platform to simultaneously detect the activity of MMP-2, MMP-9, as well as HA-ase in the urine of bladder cancer patients being monitored for disease progression.